Proteomics, toxicity and antivenom neutralization of Sri Lankan and Indian Russell's viper (Daboia russelii) venoms.

BACKGROUND: The western Russell's viper (Daboia russelii) is widely distributed in South Asia, and geographical venom variation is anticipated among distant populations. Antivenoms used for Russell's viper envenomation are, however, raised typically against snakes from Southern India. The present study investigated and compared the venom proteomes of D. russelii from Sri Lanka (DrSL) and India (DrI), the immunorecognition of Indian VINS Polyvalent Antivenom (VPAV) and its efficacy in neutralizing the venom toxicity. METHODS: The venoms of DrSL and DrI were decomplexed with C18 high-performance liquid chromatography and SDS-polyacrylamide gel electrophoresis under reducing conditions. The proteins fractionated were identified through nano-ESI-liquid chromatography-tandem mass spectrometry (LCMS/MS). The immunological studies were conducted with enzyme-linked immunosorbent assay. The neutralization of the venom procoagulant effect was evaluated in citrated human plasma. The neutralization of the venom lethality was assessed in vivo in mice adopting the WHO protocol. RESULTS: DrSL and DrI venom proteomes showed comparable major protein families, with phospholipases A2 (PLA2) being the most abundant (> 60% of total venom proteins) and diverse (six protein forms identified). Both venoms were highly procoagulant and lethal (intravenous median lethal dose in mice, LD50 = 0.24 and 0.32 µg/g, for DrSL and DrI, respectively), while lacking hemorrhagic and anticoagulant activities. VPAV was immunoreactive toward DrSL and DrI venoms, indicating conserved protein antigenicity in the venoms. The high molecular weight venom proteins were, however, more effectively immunorecognized than small ones. VPAV was able to neutralize the coagulopathic and lethal effects of the venoms moderately. CONCLUSION: Considering that a large amount of venom can be injected by Russell's viper during envenomation, the potency of antivenom can be further improved for optimal neutralization and effective treatment. Region-specific venoms and key toxins may be incorporated into the immunization procedure during antivenom production.

Effectiveness of an intervention to increase the knowledge, attitude, and practice regarding the return and disposal of unused medications.

This study aimed to evaluate the effectiveness of an educational intervention (Safe D.U.M.P) to improve the knowledge, attitude, and practice regarding the return and disposal of unused medications. Community-dwelling adults in Malaysia who could understand English were recruited from two healthcare events. Participants were asked to fill out the validated Return and Disposal of Unused Medications (ReDiUM) questionnaire (pre-intervention), view six educational intervention posters on how to dispose of unused medications (Safe D.U.M.P), then answer the ReDiUM questionnaire immediately after viewing the posters (post-intervention). A total of 390 out of 456 participants participated (response rate=85.5%). Most were female (71%) with a median age of 42 years. The overall knowledge of participants significantly increased from 60% to 80% (p<0.001). However, no improvement was seen regarding their overall attitude and practice. This outcome was as expected as it may be more difficult to improve attitude and practice (when compared to knowledge) with a single educational session.

Proteomics, toxicity and antivenom neutralization of Sri Lankan and Indian Russell's viper (Daboia russelii) venoms

The western Russell's viper (Daboia russelii) is widely distributed in South Asia, and geographical venom variation is anticipated among distant populations. Antivenoms used for Russell's viper envenomation are, however, raised typically against snakes from Southern India. The present study investigated and compared the venom proteomes of D. russelii from Sri Lanka (DrSL) and India (DrI), the immunorecognition of Indian VINS Polyvalent Antivenom (VPAV) and its efficacy in neutralizing the venom toxicity. Methods: The venoms of DrSL and DrI were decomplexed with C18 high-performance liquid chromatography and SDS-polyacrylamide gel electrophoresis under reducing conditions. The proteins fractionated were identified through nano-ESI-liquid chromatography-tandem mass spectrometry (LCMS/MS). The immunological studies were conducted with enzyme-linked immunosorbent assay. The neutralization of the venom procoagulant effect was evaluated in citrated human plasma. The neutralization of the venom lethality was assessed in vivo in mice adopting the WHO protocol. Results: DrSL and DrI venom proteomes showed comparable major protein families, with phospholipases A2 (PLA2) being the most abundant (> 60% of total venom proteins) and diverse (six protein forms identified). Both venoms were highly procoagulant and lethal (intravenous median lethal dose in mice, LD50 = 0.24 and 0.32 µg/g, for DrSL and DrI, respectively), while lacking hemorrhagic and anticoagulant activities. VPAV was immunoreactive toward DrSL and DrI venoms, indicating conserved protein antigenicity in the venoms. The high molecular weight venom proteins were, however, more effectively immunorecognized than small ones. VPAV was able to neutralize the coagulopathic and lethal effects of the venoms moderately. Conclusion: Considering that a large amount of venom can be injected by Russell's viper during envenomation, the potency of antivenom can be further improved for optimal neutralization and effective treatment. Region-specific venoms and key toxins may be incorporated into the immunization procedure during antivenom production.(AU)

Acceptance of interprofessional learning between medical and pharmacy students in a prescribing skills training workshop: pre-post intervention study.

BACKGROUND: The success of interprofessional collaboration in healthcare services requires a paradigm shift in the training of future health profession practitioners. This study aimed to develop and validate an instrument to measure Student Acceptance of Interprofessional Learning (SAIL) in Malaysia, and to assess this attribute among medical and pharmacy students using a prescribing skills training workshop. METHODS: The study consisted of two phases. In Phase 1, a 10-item instrument (SAIL-10) was developed and tested on a cohort of medical and pharmacy students who attended the workshop. In Phase 2, different cohorts of medical and pharmacy students completed SAIL-10 before and after participating in the workshop. RESULTS: Factor analysis showed that SAIL-10 has two domains: "facilitators of interprofessional learning" and "acceptance to learning in groups". The overall SAIL-10 and the two domains have adequate internal consistency and stable reliability. The total score and scores for the two domains were significantly higher after students attended the prescribing skills workshop. CONCLUSIONS: This study produced a valid and reliable instrument, SAIL-10 which was used to demonstrate that the prescribing skills workshop, where medical and pharmacy students were placed in an authentic context, was a promising activity to promote interprofessional learning among future healthcare professionals.

Evaluating the physicochemical properties and efficacy of recently expired and aged antivenom products from Thailand and Taiwan.

Gel filtration chromatography and gel electrophoresis revealed minimal protein degradation in lyophilized antivenoms which were 2-year expired (Hemato Polyvalent, Neuro Polyvalent; Thailand) and 18-year expired (Hemato Bivalent, Neuro Bivalent; Taiwan). All expired antivenoms retained immunological binding activity, and were able to neutralize the hemotoxic or neurotoxic as well as lethal effects of the homologous snake venoms. The findings show that antivenoms under proper storage conditions may remain relatively stable beyond the indicated shelf life.

Venom Proteome of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia: Toxicity Correlation, Immunoprofiling and Cross-Neutralization by Sea Snake Antivenom.

The venom proteome of Hydrophis curtus (synonym: Lapemis hardwickii) from Penang, Malaysia was investigated with nano-electrospray ionization-liquid chromatography tandem mass spectrometry (ESI-LCMS/MS) of the reverse-phase high-performance liquid chromatography (HPLC) venom fractions. Thirty distinct protein forms were identified as toxins from ten families. The three major protein families were phospholipase A2 (PLA2, 62.0% of total venom proteins), three-finger toxin (3FTX, 26.33%) and cysteine-rich secretory protein (CRiSP, 9.00%). PLA2 comprises diverse homologues (11 forms), predominantly the acidic subtypes (48.26%). 3FTX composed of one short alpha-neurotoxin (SNTX, 22.89%) and four long alpha-neurotoxins (LNTX, 3.44%). Both SNTX and LNTX were lethal in mice (intravenous LD50 = 0.10 and 0.24 µg/g, respectively) but the PLA2 were non-lethal (LD50 >1 µg/g). The more abundant and toxic SNTX appeared to be the main driver of venom lethality (holovenom LD50 = 0.20 µg/g). The heterologous Sea Snake Antivenom (SSAV, Australia) effectively cross-neutralized the venom (normalized potency = 9.35 mg venom neutralized per g antivenom) and the two neurotoxins in vivo, with the LNTX being neutralized more effectively (normalized potency = 3.5 mg toxin/g antivenom) than SNTX (normalized potency = 1.57 mg/g). SSAV immunorecognition was strong toward PLA2 but moderate-to-weak toward the alpha-neurotoxins, indicating that neutralization of the alpha-neurotoxins should be further improved.

Development and Validation of the Return and Disposal of Unused Medications Questionnaire (ReDiUM) in Malaysia.

The aim of our study was to develop and validate the return and disposal of unused medications (ReDiUM), as there are currently no validated tools available (worldwide) to assess this. The ReDiUM was developed by an expert panel. It was then administered to community-dwelling adults, who could understand English, at baseline and 2 weeks later. A total of 338/354 participants agreed to participate (response rate = 95.4%). Flesch reading ease was 60. The overall Cronbach's α was 0.703 (range = 0.609-0.762). At test-retest, κ values ranged from 0.244 to 0.523. Median total knowledge score was 60% (interquartile range = 40% to 70%). The majority of participants (94.4%) knew that improper drug disposal has harmful effects on the environment. However, their knowledge was low (11.2%) regarding the disposal of pressurized metered-dose inhalers in the garbage. The ReDiUM was found to be a valid and reliable instrument to assess the knowledge, attitude, and practice on the ReDiUM in Malaysia.

Proteomics, functional characterization and antivenom neutralization of the venom of Pakistani Russell's viper (Daboia russelii) from the wild.

The venom proteome of wild Pakistani Russell's viper (Daboia russelii) was investigated through nano-ESI-LCMS/MS of the reverse-phase HPLC fractions. A total of 54 venom proteins were identified and clustered into 11 protein families. Phospholipase A2 (PLA2, 63.8%) and Kunitz-type serine protease inhibitor (KSPI, 16.0%) were most abundant, followed by snake venom serine protease (SVSP, 5.5%, mainly Factor V activating enzyme), vascular endothelial growth factor (VEGF, 4.3%), snake venom metalloproteinase (SVMP, 2.5%, mainly Factor X activating enzyme) and phosphodiesterase (PDE, 2.5%). Other minor proteins include cysteine-rich secretory protein (CRiSP), snake venom C-type lectin/lectin-like protein (snaclec), nerve growth factor, L-amino acid oxidase and 5'-nucleotidase. PLA2, KSPI, SVSP, snaclec and SVMP are hemotoxic proteins in the venom. The study indicated substantial venom variation in D. russelii venoms of different locales, including 3 Pakistani specimens kept in the USA. The venom exhibited potent procoagulant activity on human plasma (minimum clotting dose = 14.5 ng/ml) and high lethality (rodent LD50 = 0.19 µg/g) but lacked hemorrhagic effect locally. The Indian VINS Polyvalent Antivenom bound the venom immunologically in a concentration-dependent manner. It moderately neutralized the venom procoagulant and lethal effects (normalized potency against lethality = 2.7 mg venom neutralized per g antivenom). BIOLOGICAL SIGNIFICANCE: Comprehensive venom proteomes of D. russelii from different locales will facilitate better understanding of the geographical variability of the venom in both qualitative and quantitative terms. This is essential to provide scientific basis for the interpretation of differences in the clinical presentation of Russell's viper envenomation. The study revealed a unique venom proteome of the Pakistani D. russelii from the wild (Indus Delta), in which PLA2 predominated (~60% of total venom proteins). The finding unveiled remarkable differences in the venom compositions between the wild (present study) and the captive specimens reported previously. The integration of toxicity tests enabled the correlation of the venom proteome with the envenoming pathophysiology, where the venom showed potent lethality mediated through coagulopathic activity. The Indian VINS Polyvalent Antivenom (VPAV) showed binding activity toward the venom protein antigens; however the immunorecognition of small proteins and PLA2-dominating fractions was low to moderate. Consistently, the antivenom neutralized the toxicity of the wild Pakistani Russell's viper venom at moderate efficacies. Our results suggest that it may be possible to enhance the Indian antivenom potency against the Pakistani viper venom by the inclusion of venoms from a wider geographical range including that from Pakistan into the immunogen formulation.

Geographical venom variations of the Southeast Asian monocled cobra (Naja kaouthia): venom-induced neuromuscular depression and antivenom neutralization.

The Southeast Asian monocled cobras (Naja kaouthia) exhibit geographical variations in their venom proteomes, especially on the composition of neurotoxins. This study compared the neuromuscular depressant activity of the venoms of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V), and the neutralization of neurotoxicity by a monospecific antivenom. On chick biventer cervicis nerve-muscle preparation, all venoms abolished the indirect twitches, with NK-T venom being the most potent (shortest t90, time to 90% twitch inhibition), followed by NK-V and NK-M. Acetylcholine and carbachol failed to reverse the blockade, indicating irreversible/pseudo-irreversible post-synaptic neuromuscular blockade. KCl restored the twitches variably (NK-M preparation being the least responsive), consistent with different degree of muscle damage. The findings support that NK-T venom has the most abundant curarimimetic alpha-neurotoxins, while NK-M venom contains more tissue-damaging cytotoxins. Pre-incubation of tissue with N. kaouthia monovalent antivenom (NKMAV) prevented venom-induced twitch depression, with the NK-T preparation needing the largest antivenom dose. NKMAV added after the onset of neuromuscular depression could only halt the inhibitory progression but failed to restore full contraction. The findings highlight the urgency of early antivenom administration to sequester as much circulating neurotoxins as possible, thereby hastening toxin elimination from the circulation. In envenomed mice, NKMAV administered upon the first neurological sign neutralized the neurotoxic effect, with the slowest full recovery noticed in the NK-T group. This is consistent with the high abundance of neurotoxins in the NK-T venom, implying that a larger amount or repeated dosing of NKMAV may be required in NK-T envenomation.

Development and validation of an instrument to assess the prescribing readiness of medical students in Malaysia.

BACKGROUND: Prescribing incompetence is an important factor that contributes to prescribing error, and this is often due to inadequate training during medical schools. We therefore aimed to develop and validate an instrument to assess the prescribing readiness of medical students (PROMS) in Malaysia. METHODS: The PROMS comprised of 26 items with four domains: undergraduate learning opportunities; hands-on clinical skills practice; information gathering behaviour; and factors affecting the learning of prescribing skills. The first three domains were adapted from an existing questionnaire, while items from the last domain were formulated based on findings from a nominal group discussion. Face and content validity was determined by an expert panel, pilot tested in a class of final year (Year 5) medical students, and assessed using the Flesch reading ease. To assess the reliability of the PROMS, the internal consistency and test-retest (at baseline and 2 weeks later) were assessed using the Wilcoxon Signed Ranks test and Spearman's rho. The discriminative validity of the PROMS was assessed using the Mann-Whitney U-test (to assess if the PROMS could discriminate between final year medical students from a public and a private university). RESULTS: A total of 119 medical students were recruited. Flesch reading ease was 46.9, indicating that the instrument was suitable for use in participants undergoing tertiary education. The overall Cronbach alpha value of the PROMS was 0.695, which was satisfactory. Test-retest showed no difference for 25/26 items, indicating that our instrument was reliable. Responses from the public and private university final year medical students were significantly different in 10/26 items, indicating that the PROMS was able to discriminate between these two groups. Medical students from the private university reported fewer learning opportunities and hands-on practice compared to those from the public university. On the other hand, medical students from the private university reported more frequent use of both web based and non-web-based resources compared to their public university counterparts. CONCLUSIONS: The PROMS instrument was found to be a reliable and valid tool for assessing medical students' readiness to prescribe in Malaysia. It may also inform on the adequacy of medical programmes in training prescribing skills.

Immunological cross-reactivity and neutralization of the principal toxins of Naja sumatrana and related cobra venoms by a Thai polyvalent antivenom (Neuro Polyvalent Snake Antivenom).

The low potency of cobra antivenom has been an area of concern in immunotherapy for cobra envenomation. This study sought to investigate factors limiting the neutralizing potency of cobra antivenom, using a murine model. We examined the immunological reactivity and neutralizing potency of a Thai polyvalent antivenom against the principal toxins of Naja sumatrana (Equatorial spitting cobra) venom and two related Asiatic cobra venom α-neurotoxins. The antivenom possesses moderate neutralizing potency against phospholipases A2 (P, potency of 0.98mg/mL) and moderately weak neutralizing potency against long-chain α-neurotoxins (0.26-0.42mg/mL) but was only weakly effective in neutralizing the short-chain α-neurotoxins and cardiotoxins (0.05-0.08mg/mL). The poor neutralizing potency of the antivenom on the low molecular mass short-chain neurotoxins and cardiotoxins is presumably the main limiting factor of the efficacy of the cobra antivenom. Our results also showed that phospholipase A2, which exhibited the highest ELISA reactivity and avidity, was most effectively neutralized, whereas N. sumatrana short-chain neurotoxin, which exhibited the lowest ELISA reactivity and avidity, was least effectively neutralized by the antivenom. These observations suggest that low immunoreactivity (low ELISA reactivity and avidity) is one of the reasons for poor neutralization of the cobra venom low molecular mass toxins. Nevertheless, the overall results show that there is a lack of congruence between the immunological reactivity of the toxins toward antivenom and the effectiveness of toxin neutralization by the antivenom, indicating that there are other factors that also contribute to the weak neutralization capacity of the antivenom. Several suggestions have been put forward to overcome the low efficacy of the cobra antivenom. The use of a 'proper-mix' formulation of cobra venoms as immunogen, whereby the immunogen mixture used for hyperimmunization contains a mix of various types of α-neurotoxins and cardiotoxins in sufficient amount, may also help to improve the efficacy and broaden the neutralization spectrum of the antivenom.

The Effect of a Polyvalent Antivenom on the Serum Venom Antigen Levels of Naja sputatrix (Javan Spitting Cobra) Venom in Experimentally Envenomed Rabbits.

The treatment protocol of antivenom in snake envenomation remains largely empirical, partly due to the insufficient knowledge of the pharmacokinetics of snake venoms and the effects of antivenoms on the blood venom levels in victims. In this study, we investigated the effect of a polyvalent antivenom on the serum venom antigen levels of Naja sputatrix (Javan spitting cobra) venom in experimentally envenomed rabbits. Intravenous infusion of 4 ml of Neuro Polyvalent Snake Antivenom [NPAV, F(ab')2 ] at 1 hr after envenomation caused a sharp decline of the serum venom antigen levels, followed by transient resurgence an hour later. The venom antigen resurgence was unlikely to be due to the mismatch of pharmacokinetics between the F(ab')2 and venom antigens, as the terminal half-life and volume of distribution of the F(ab')2 in serum were comparable to that of venom antigens (p > 0.05). Infusion of an additional 2 ml of NPAV was able to prevent resurgence of the serum venom antigen level, resulting in a substantial decrease (67.1%) of the total amount of circulating venom antigens over time course of envenomation. Our results showed that the neutralization potency of NPAV determined by neutralization assay in mice may not be an adequate indicator of its capability to modulate venom kinetics in relation to its in vivo efficacy to neutralize venom toxicity. The findings also support the recommendation of giving high initial dose of NPAV in cobra envenomation, with repeated doses as clinically indicated in the presence of rebound antigenemia and symptom recurrence.

Proteomic investigation of Sri Lankan hump-nosed pit viper (Hypnale hypnale) venom.

The hump-nosed pit viper, Hypanle hypnale, contributes to snakebite mortality and morbidity in Sri Lanka. Studies showed that the venom is hemotoxic and nephrotoxic, with some biochemical and antigenic properties similar to the venom of Calloselasma rhodostoma (Malayan pit viper). To further characterize the complexity composition of the venom, we investigated the proteome of a pooled venom sample from >10 Sri Lankan H. hypnale with reverse-phase high performance liquid chromatography (rp-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and peptide sequencing (tandem mass-spectrometry and/or N-terminal sequencing). The findings ascertained that two phospholipase A2 subtypes (E6-PLA2, W6-PLA2) dominate the toxin composition by 40.1%, followed by snake venom metalloproteases (36.9%), l-amino acid oxidase (11.9%), C-type lectins (5.5%), serine proteases (3.3%) and others (2.3%). The presence of the major toxins correlates with the venom's major pathogenic effects, indicating these to be the principal target toxins for antivenom neutralization. This study supports the previous finding of PLA2 dominance in the venom but diverges from the view that H. hypnale venom has low expression of large enzymatic toxins. The knowledge of the composition and abundance of toxins is essential to elucidate the pathophysiology of H. hypnale envenomation and to optimize antivenom formulation in the future.

Prophylactic effect of Mucuna pruriens Linn (velvet bean) seed extract against experimental Naja sputatrix envenomation: gene expression studies.

Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.

Anti-inflammatory effect of the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden, the Tiger Milk mushroom.

BACKGROUND: The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) is used as a traditional medicine to relieve cough, asthma and chronic hepatitis. The traditional uses of the sclerotium are presumably related to its anti-inflammatory effect. The present study was carried out to evaluate the anti-inflammatory activity of the sclerotial powder of L. rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) cultivar TM02. METHODS: The anti-acute inflammatory activity of the sclerotial powder of L. rhinocerotis cultivar TM02 was investigated using carrageenan-induced paw edema test while the inhibition of transudative and proliferative phases of chronic inflammation were studied by cotton pellet induced granuloma model. Sprague Dawley rats were used in both studies. The anti-inflammatory activity was also measured by inhibition of lipopolysaccharide induced TNF-alpha production in RAW 264.7 macrophage cells. RESULTS: Cold water extract (CWE), hot water extract (HWE) and methanol extract (ME) of the sclerotial powder of L. rhinocerotis cultivar TM02 possessed anti-acute inflammatory activity as was measured by carrageenan-induced paw edema test, with CWE being the most potent. The acute anti-inflammatory activity of the cold water extract (CWE) was mainly contributed by its high molecular weight (HMW) fraction isolated by Sephadex G50 gel filtration chromatography. Its protein component was very potent in the inhibition of TNF-alpha production with an IC50 of 0.76 µg/ml. CWE at 200 mg/kg did not inhibit transudative and proliferative phase of chronic inflammation as shown by using the cotton pellet induced granuloma model. CONCLUSIONS: Our results suggested that most of the bioactive substance(s) contributed to the acute-inflammatory activity of the sclerotial powder of L. rhinocerotis cultivar TM02 appear to be in the CWE, particularly its HMW fraction. The anti-inflammatory activity of CWE was mainly contributed by the protein component of the high molecular weight (HMW) fraction and it exhibited strong inhibitory effect on TNF-alpha production but the possibility of synergistic effect between HMW, MMW and LMW fractions cannot be excluded. Future studies will provide new insights into the anti-inflammatory activity of the sclerotial powder.

Pharmacokinetics of Naja sumatrana (equatorial spitting cobra) venom and its major toxins in experimentally envenomed rabbits.

BACKGROUND: The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom's composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A2, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits. PRINCIPAL FINDINGS: The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A2 and whole venom (Tmax=2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (Fi.m.=81.5%) compared to that of the phospholipase A2 (Fi.m.=68.6%) or cardiotoxin (Fi.m.=45.6%). The incomplete absorption of the phospholipase A2 and cardiotoxin may infer the toxins' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions. CONCLUSION/SIGNIFICANCE: Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.

Pharmacokinetics of the Sri Lankan hump-nosed pit viper (Hypnale hypnale) venom following intravenous and intramuscular injections of the venom into rabbits.

The knowledge of venom pharmacokinetics is essential to improve the understanding of envenomation pathophysiology. Using a double-sandwich ELISA, this study investigated the pharmacokinetics of the venom of hump-nosed pit viper (Hypnale hypnale) following intravenous and intramuscular injections into rabbits. The pharmacokinetics of the venom injected intravenously fitted a three-compartment model. There is a rapid (t1/2π = 0.4 h) and a slow (t1/2α = 0.8 h) distribution phase, followed by a long elimination phase (t1/2ß = 19.3 h) with a systemic clearance of 6.8 mL h(-1) kg(-1), consistent with the prolonged abnormal hemostasis reported in H. hypnale envenomation. On intramuscular route, multiple peak concentrations observed in the beginning implied a more complex venom absorption and/or distribution pattern. The terminal half-life, volume of distribution by area and systemic clearance of the venom injected intramuscularly were nevertheless not significantly different (p > 0.05) from that of the venom injected intravenously. The intramuscular bioavailability was exceptionally low (Fi.m. = 4%), accountable for the highly varied median lethal doses between intravenous and intramuscular envenomations in animals. The findings indicate that the intramuscular route of administration does not significantly alter the pharmacokinetics of H. hypnale venom although it significantly reduces the systemic bioavailability of the venom.

Cross neutralization of common Southeast Asian viperid venoms by a Thai polyvalent snake antivenom (Hemato Polyvalent Snake Antivenom).

Snake envenomation is a serious public health threat in many rural areas of Asia and Africa. Antivenom has hitherto been the definite treatment for snake envenomation. Owing to a lack of local production of specific antivenom, most countries in these regions fully depend on foreign supplies of antivenoms. Often, the effectiveness of the imported antivenoms against local medically important species has not been validated. This study aimed to assess cross-neutralizing capacity of a recently developed polyvalent antivenom, Hemato Polyvalent Snake Antivenom (HPAV), against venoms of a common viper and some pit vipers from Southeast Asia. Neutralisation assays showed that HPAV was able to effectively neutralize lethality of the common Southeast Asian viperid venoms examined (Calloselasma, Crytelytrops, Popeia, and Daboia sp.) except for Tropidolaemus wagleri venom. HPAV also effectively neutralized the procoagulant and hemorrhagic activities of all the venoms examined, corroboratively supporting the capability of HPAV in neutralizing viperid venoms which are principally hematoxic. The study also indicated that HPAV fully prevented the occurrence of hematuria and proteinuria in mice envenomed with Thai Daboia siamensis venom but was only partially effective against venoms of Myanmar D. siamensis. Thus, HPAV appears to be useful against its homologous venoms and venoms from Southeast Asian viperids including several medically important pit vipers belonging to the Trimeresurus complex. Nevertheless, the effectiveness of HPAV as a paraspecific antivenom for treatment of viperid envenomation in Southeast Asian region requires further assessment from future clinical trials.

Toxicokinetics of Naja sputatrix (Javan spitting cobra) venom following intramuscular and intravenous administrations of the venom into rabbits.

Existing protocols for antivenom treatment of snake envenomations are generally not well optimized due partly to inadequate knowledge of the toxicokinetics of venoms. The toxicokinetics of Naja sputatrix (Javan spitting cobra) venom was investigated following intravenous and intramuscular injections of the venom into rabbits using double-sandwich ELISA. The toxicokinetics of the venom injected intravenously fitted a two-compartment model. When the venom was injected intramuscularly, the serum concentration-time profile exhibited a more complex absorption and/or distribution pattern. Nevertheless, the terminal half-life, volume of distribution by area and systemic clearance of the venom injected intramuscularly were not significantly different (p > 0.05) from that of the venom injected intravenously. The systemic bioavailability of the venom antigens injected by intramuscular route was 41.7%. Our toxicokinetic finding is consistent with other reports, and may indicate that some cobra venom toxins have high affinity for the tissues at the site of injection. Our results suggest that the intramuscular route of administration doesn't significantly alter the toxicokinetics of N. sputatrix venom although it significantly reduces the systemic bioavailability of the venom.